Use of a Sec-dependent secretion system for secreting proteins that are usually secreted by a Sec-independent secretion system, bacteria containing it and their use

ABSTRACT

The invention relates to the use of a Sec-dependent secretion system for secreting proteins normally secreted by a Sec-independent secretion system.

The present invention relates to the use of a Sec-dependent secretionsystem for secreting proteins normally secreted by a Sec-independentsecretion system. It further relates to the lactic acid bacteriacontaining this Sec-dependent secretion system, in particular thelactococci belonging to the species Lactococcus lactis, to the use ofcertain strains of these lactococci for transferring said secretionsystem to strains of industrial interest, particularly in the dairyindustry, and to the use of certain strains of Lactococcus lactis forobtaining this secretion system.

The secretion of proteins in bacteria generally follows two pathways.The better known is Sec-dependent transport. According to this pathway,the protein is exported by virtue of the presence, in the N-terminalposition, of a signal peptide which is cleaved in the secreted protein.Several proteins involved in this secretion, particularly SecA, SecY andSecE, have been identified in E. coli. The secY gene has been identifiedin L. lactis: it possesses a high degree of homology with the secY geneof E. coli, indicating that this secretion system is very probably alsopresent in L. lactis (Koivula et al., 1991, FEBS Lett. 228: 114-118).

The second secretion pathway in bacteria, which does not depend on theSec proteins, involves transmembrane translocators called the A, B, Cfamily of proteins (Pugsley, 1993, Microbiol. Rev. 57: 50-108).

Translocators have been identified in L. lactis; they are involved inthe secretion of the bacteriocins LcnA, LcnB and LcnMl and theantibiotics lacticin 481, nisin A and nisin Z (de Vos and Simons,Genetics and Biotechnology of L.A.B., Blackie Academic and Professional,1994).

The study of these two secretion pathways in L. lactis has been utilizedto develop different systems for the secretion of heterologous proteins.The secretion vectors developed are all contained in the review by deVos and Simons, Genetics and Biotechnology of L.A.B., Blackie Academicand Professional, 1994.

The bacteriocins identified in lactic acid bacteria are virtually allsecreted by the Sec-independent system (Dodd and Gasson, Genetics andBiotechnology of L.A.B., Blackie Academic and Professional, 1994).However, in the interest of increasing the secretion of thesebacteriocins, it can be important to cause secretion of thesebacteriocins by a different secretion system.

Some authors have recently shown that divergicin A, a bacteriocinproduced by Carnobacterium divergens, is secreted via a Sec-dependentsystem (Worobo et al., J. Bacteriol. 177: 3143-3149). However, it hasnever been shown in lactic acid bacteria that a bacteriocin secreted bya Sec-independent system can be secreted by a Sec-dependent system.

The Applicant has carried out studies in this field and has found,surprisingly, that a Sec-dependent secretion system can be used tosecrete proteins normally secreted by a Sec-independent secretionsystem.

According to the invention, the Sec-dependent secretion system is usedin combination with the DNA sequence coding for a mature proteinnormally secreted by a Sec-independent system, and with an appropriatepromoter, an appropriate signal sequence recognized by the Sec-dependentsecretion system and an appropriate terminator.

Thus the invention further relates to the DNA constructions for thesecretion, by a Sec-dependent system, of proteins normally secreted by aSec-independent system, said DNA constructions comprising a promoter, asignal sequence recognized by the Sec-dependent secretion system, theDNA sequence coding for a mature protein normally secreted by aSec-independent system, and a terminator. These DNA constructions can bepresent on an expression vector such as a plasmid, in the genomic DNA orin any other DNA fragment.

According to the invention, any Sec-dependent secretion system can beused in combination with a gene coding for a protein of interest whichis normally secreted by a Sec-independent system.

Any Sec-dependent secretion system is suitable for the purposes of theinvention. Particularly appropriate Sec-dependent secretion systems arethe Sec-dependent systems of lactococci, which utilize in particular thesp signal sequence of the protease of lactococcus (PrtP protein).

Any promoters and terminators which are compatible with the host strainin which the secretion system of the invention is used can be employedaccording to the invention.

As examples of genes coding for a protein of interest, it is possible touse especially the genes coding for bacteriocins, in particular thebacteriocin LcnB, which is a bacteriocin secreted by L. lactis under thedependence of a Sec-independent secretion system (Van Belkum et al.,1992, Applied Environ. Microbiol., 58: 572-577).

Thus, in one variant, the invention relates to the expression vectorscomprising a promoter, a signal sequence recognized by the Sec-dependentsecretion system, the DNA sequence coding for a bacteriocin such as thebacteriocin LcnB, and a terminator.

In this variant, the promoter can advantageously be the P32 promoter oflactococcus, the signal sequence can be the sp signal sequence of theprotease (PrtP) of lactococcus and the terminator can advantageously bethat of the LcnB immunity gene (ciB).

The invention further relates to the lactic acid bacteria, preferably L.lactis, secreting one or more bacteriocins, which contain a constructionaccording to the invention, as defined above, which permits thesecretion of these bacteriocins. This construction can be introducedinto the lactic acid bacteria by conjugation, transformation, protoplastfusion or another gene transfer method.

Examples of the lactic acid bacteria which can advantageously betransformed with the aid of the construction according to the inventionare Lactococcus lactis ssp crenioris, Lactococcus lactis ssp lactis andlactococcus lactis ssp lactis var diacetylactis.

These strains transformed in this way can be used to transfer aconstruction according to the invention to a strain of industrialinterest by conjugation, transformation, transduction, protoplast fusionor another gene transfer method. This construction can be carried by aplasmid or by another part of the bacterial genome.

The invention further relates to the strains of industrial interestwhich secrete proteins obtained in this way, for example bacteriocins.These bacteria which secrete bacteriocins can be used to inhibit thedevelopment of pathogenic bacteria, to lyze bacteria so that theirenzyme content participates in the purification of the productsfermented by these bacteria, or to have bacteria for any purposeinvolving a secretion of bacteriocins by a construction according to theinvention.

The invention will be understood more clearly with the aid of theillustrative Examples below, which do not imply a limitation.

The bulk of the techniques described in these Examples, which are wellknown to those skilled in the art, are described in detail in the workby Sambrook, Fritsch and Maniatis: "Molecular cloning; a laboratorymanual", published in 1989 by Cold Spring Harbor Press, N.Y. (2ndedition).

The following description will be understood more clearly with the aidof FIGS. 1 to 3 below, which respectively show:

FIG. 1: Construction of vector pSV123

FIG. 2: Construction of secretion vector pSV

FIG. 3: Construction of plasmid pSVLcnB permitting the secretion of LcnB

EXAMPLE 1: CONSTRUCTION OF PLASMID SECRETION VECTOR PSV

To be able to secrete a bacteriocin whose secretion is Sec-independent,it was necessary first to construct a Sec-dependent secretion vector.This construction was prepared in the following manner:

For each of the clonings where is was necessary to do so, the PCRtechnique was used under the following conditions: 1 minute at 94° C.(denaturation), 2 minutes at 45° C. (hybridization) and 2 minutes at 73°C. (polymerization), 30 cycles. The oligonucleotides were synthesizedwith an Applied Biosystems 381A DNA synthesizer (Applied Biosystems).

Oligonucleotides A, B, C and D were used to construct secretion vectorpSV.

The sequences of these oligonucleotides are as follows (the restrictionsites used for cloning are underlined and the RBS in oligonucleotide Bis emboldened):

    Oligonucleotide A (SEQ ID No 1):         BglII BglII EcoRI    5'-AGATCTAGATCTGAATTCGGTCCTCGGGATAATG-3'    Oligonucleotide B (SEQ ID No 2):          PstI PstI   ClaI      RBS    5'-CTGCAGCTGCAGCATCGATAAATTCCTCCGAATATTTTTTTACC-3'    Oligonucleotide C (SEQ ID No 3):          BglII BglII  ClaI    5'-AGATCTAGATCTATCGATGCAAAGGAAAAAGAAAGGGCTTTCGATCTTG-3'40    Oligonucleotide D (SEQ ID No 4):         PstI PstI SacI SacII    5'-CTGCAGCTGCAGAGCTCGCCGCGGCCTTTGCTTGGATTTCGCCG-3'

PCR (polymerase chain reaction) products containing the promoter and theribosome binding site, RBS, of ORF32 (open reading frame) were obtainedfrom plasmid pMG36c (Van de Guchte et al., 1989, Applied Environ.Microbiol., 55: 224-228), which is derived from plasmid pMG36e usedbelow.

The sp signal sequence of the protease of lactococcus, PrtP, wasobtained from the prtP gene present in plasmid pGKV500 (Kok et al.,1988, Applied Environ. Microbiol., 54: 221-228).

After digestion with the restriction enzymes BgIII and PstI, these PCRproducts were cloned into plasmid pUK21 (Vieira and Messing, 1991, Gene,100: 189-194) to give plasmids pSVI and pSV23 (FIG. 1). Plasmid pSV23was then digested with the restriction enzymes to ClaI and NdeI and thefragment containing the signal sequence was cloned into plasmid pSVI,previously digested with the restriction enzymes ClaI and NdeI, to giveplasmid pSV123 (FIG. 1). Finally, the EcoRI-SacI fragment of pSVI23,containing the P32 promoter and the sp signal sequence, was cloned intoplasmid pMG36e (Van de Guchte et al., 1989, Applied Environ. Microbiol.,SS: 224-228) to give secretion vector pSV (FIG. 2).

Example 2: Construction of plasmid pSVLcnB involved in the secretion ofthe bacteriocin LcnB

Plasmid pMB580 (a plasmid conferring erythromycin resistance, derivedfrom plasmid pGK210 and containing the operon of lactococcin B, i.e.LcnB and lciB - Van Belkum et al., 1992, Applied Environ. Microbiol.,58: 572-577) was digested with the restriction enzymes to NsiI andHindIII. The Hindlll end was hydrolyzed to convert it to a blunt end.The HindllI-NsiI fragment was ligated to plasmid pSV, which hadpreviously been digested with the restriction enzymes to PstI and Saclland whose Sacll end had been hydrolyzed to convert it to a blunt end, togive plasmid pSVLcnB (FIG. 3). The resulting amino acid sequence of thisLcnB derivative with the signal sequence of the PrtP protein is givenbelow (SEQ ID N° 5):

N-MQRKKKGLSILLAGTVALGALAVLPVGEIQAKA

SLQYVMSAGPYTWYKDERTGKTECKQTIDTASYTFGVMAEGWGKTFH-C

Example 3: Secretion of LcnB by a Sec-dependent secretion system

The plasmid derived from plasmid pSV (pSVLcnB), containing thestructural gene of lactococcin LcnB (lcnB) and the LcnB immunity gene(lcnB), was used to transform the strain E. coli JM101 (Maniatis et al.,1982, Molecular Cloning: a laboratory manual. Cold Spring Harbor, N.Y.,Cold Spring Harbor Laboratory), the strain Lactococcus lactis MG1363(Gasson, 1983, J. Bacteriol., 154: 1-9) and the strain Lactococcuslactis IL1403 (Chopin et al., 1984, Plasmid, 11: 260-263). In theseconstructions the N-terminal extension of the prebacteriocin wasreplaced with the signal peptide of PrtP (FIGS. 1, 2 and 3).

To test the bacteriocin activity, the strains were covered with anoverlayer of the strain IL1043 containing either pMG36e or pMG36c. Thistest was performed in the following manner:

Colonies containing the construction (pSVLcnB) were incubated for 16 hat 30° C. on dishes of M17 (Terzaghi and Sandine, 1975, Appl.Microbiol., 29: 807-813)+glucose 5 g/l+agar 5 g/l. After exposure tochloroform vapors for 20 minutes, the dishes were partially dried in airto remove the residual traces of chloroform. The following was thenplated out on these dishes: 4 ml of gelose M17+glucose 5 g/l+agar 7 g/linoculated with 40 μl of a night culture of the indicator strain L.lactis IL1403 containing pMG36e or pMG36c. After incubation at 30° C.for 12 to 18 h, the dishes were analyzed for the inhibition zones(halos) around the clones. The overlayer tests on tricin gels wereperformed according to the procedure described by Van Belkum et al.,1992, in Applied Environ. Microbiol., 58: 572-577.

The results showed that there is a halo of inhibition around the clonescontaining pSVLcnB. This halo is wider around the clones containingpSVLcnBc. This indicates that the bacteriocin LcnB was indeed secretedby virtue of pSVLcnB and pSVLcnBc. To confirm this result and inparticular to know whether the bacteriocin activity is due to a maturedform (without the sp signal peptide) or nonmatured form (with the spsignal peptide), culture supernatants of the strain IL1403 containingpSVLcnB were subjected to precipitation with ammonium sulfate and theprecipitate was tested on tricin SDS-PAA electrophoresis gel. The gelswere then examined for bactericidal activity. The results show thatthere is an inhibition of growth in the 3, 4 KDa zone corresponding tothe size of the matured bacteriocin LcnB, i.e. the bacteriocin fromwhich the sp signal sequence has been removed. As the strain Lactococcuslactis MG1363 does not possess the Sec-independent secretion system ofbacteriocins, the bacteriocin LcnB produced by virtue of plasmid pSVLcnBwas indeed secreted via the Sec-dependent secretion system using the spsignal sequence of the protease of lactococcus, PrtP.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 5    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 33 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: YES    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #         33       TCGG TCCTCGGGAT ATG    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 44 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: YES    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 44               GATA AATTCCTCCG AATATTTTTT TACC    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 49 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: YES    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #               49GATGC AAAGGAAAAA GAAAGGGCTT TCGATCTTG    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 44 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: YES    #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 44               CGCC GCGGCCTTTG CTTGGATTTC GCCG    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 80 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Met Gln Arg Lys Lys Lys Gly Leu Ser Ile Le - #u Leu Ala Gly Thr Val    #                15    - Ala Leu Gly Ala Leu Ala Val Leu Pro Val Gl - #y Glu Ile Gln Ala Lys    #            30    - Ala Ser Leu Gln Tyr Val Met Ser Ala Gly Pr - #o Tyr Thr Trp Tyr Lys    #        45    - Asp Glu Arg Thr Gly Lys Thr Glu Cys Lys Gl - #n Thr Ile Asp Thr Ala    #    60    - Ser Tyr Thr Phe Gly Val Met Ala Glu Gly Tr - #p Gly Lys Thr Phe His    #80    __________________________________________________________________________

We claim:
 1. A DNA construct for the secretion, by a Sec-dependentsystem, of a protein normally secreted by a Sec-independent system,characterized in that it comprises a promoter, a signal sequencerecognized by the Sec-dependent secretion system, a DNA sequence codingfor a mature protein normally secreted by a Sec-independent system, anda terminator wherein said construct does not contain a multiple cloningsite.
 2. The construct according to claim 1, characterized in that thesignal sequence recognized by the Sec-dependent secretion system is thesp signal sequence of the protease of lactococcus.
 3. The constructaccording to claim 1, characterized in that the DNA sequence coding forthe protein is the DNA sequence coding for a bacteriocin.
 4. Theconstruct according to claim 3, characterized in that the bacteriocin isthe bacteriocin LcnB.
 5. The construct according to claim 1,characterized in that the promoter is the P32 promoter of lactococcusand the terminator is that of the LcnB immunity gene (lciB).
 6. Theconstruct according to claim 2, characterized in that the DNA sequencecoding for the protein is the DNA sequence coding for a bacteriocin. 7.The construct according to claim 6, characterized in that thebacteriocin is the bacteriocin LcnB.
 8. The construct according to claim2, characterized in that the promoter is the P32 promoter of lactococcusand the terminator is that of the LcnB immunity gene (lciB).
 9. Theconstruct according to claim 3, characterized in that the promoter isthe P32 promoter of lactococcus and the terminator is that of the LcnBimmunity gene (lciB).
 10. The construct according to claim 4,characterized in that the promoter is the P32 promoter of lactococcusand the terminator is that of the LcnB immunity gene (lciB).
 11. Theconstruct according to claim 6, characterized in that the promoter isthe P32 promoter of lactococcus and the terminator is that of the LcnBimmunity gene (lciB) .
 12. The construct according to claim 7,characterized in that the promoter is the P32 promoter of lactococcusand the terminator is that of the LcnB immunity gene (lciB) .
 13. Aprocess for secreting a protein that is normally secreted by aSec-independent secretion system, using a Sec-dependent secretionsystem, comprisinga) providing a DNA sequence coding for said protein, apromoter, a signal sequence recognized by the Sec-dependent secretionsystem and a terminator; b) constructing a DNA construct comprising saidDNA sequence, said promoter, said signal sequence and said terminator;c) introducing said construct into a lactic acid bacterium by genetransfer; and d) secreting said protein by the lactic acid bacteriumcontaining said construct.
 14. The process according to claim 13,characterized in that the signal sequence is the sp signal sequence ofthe protease of lactococcus.